Department of Molecular Biology and Biochemistry, Division of Basic Life Sciences, Institute of Biomedical and Health Sciences, Hiroshima University, Hiroshima, 734-8553, Japan.
Laboratory of Cellular Differentiation, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto, 606-8507, Japan.
Sci Rep. 2017 Mar 22;7:45010. doi: 10.1038/srep45010.
Scleraxis (Scx) is a basic helix-loop-helix transcription factor that is expressed persistently in tendons/ligaments, but transiently in entheseal cartilage. In this study, we generated a novel Scx knock-in (KI) allele, by in-frame replacement of most of Scx exon 1 with Cre recombinase (Cre), to drive Cre expression using Scx promoter and to inactivate the endogenous Scx. Reflecting the intensity and duration of endogenous expression, Cre-mediated excision occurs in tendinous and ligamentous tissues persistently expressing Scx. Expression of tenomodulin, a marker of mature tenocytes and ligamentocytes, was almost absent in tendons and ligaments of Scx KI mice lacking Scx to indicate defective maturation. In homozygotes, the transiently Scx-expressing entheseal regions such as the rib cage, patella cartilage, and calcaneus were small and defective and cartilaginous tuberosity was missing. Decreased Sox9 expression and phosphorylation of Smad1/5 and Smad3 were also observed in the developing entheseal cartilage, patella, and deltoid tuberosity of Scx KI mice. These results highlighted the functional importance of both transient and persistent expression domains of Scx for proper integration of the musculoskeletal components.
腱/韧带细胞中持续表达的 Scleraxis（Scx）是一种基本螺旋-环-螺旋转录因子，但在腱/骨附着处软骨中为瞬时表达。在本研究中，我们通过 Cre 重组酶（Cre）在 Scx 外显子 1 的大部分序列上进行框内替换，生成了一种新型的 Scx 基因敲入（KI）等位基因，以 Scx 启动子驱动 Cre 表达，并使内源性 Scx 失活。Cre 介导的缺失发生在持续表达 Scx 的腱/韧带组织中，反映了内源性表达的强度和持续时间。Scx KI 小鼠缺乏 Scx，导致腱/韧带中成熟肌腱细胞和韧带细胞的标志物 tenomodulin 几乎不表达，表明成熟缺陷。在纯合子中，如肋骨、髌骨软骨和跟骨等瞬态 Scx 表达的腱/骨附着处区域较小且有缺陷，并且缺乏软骨结节。Scx KI 小鼠的发育中的腱/骨附着处软骨、髌骨和三角肌结节中 Sox9 表达减少，Smad1/5 和 Smad3 的磷酸化也减少。这些结果强调了 Scx 的瞬时和持续表达区域对于骨骼肌肉成分的正确整合的功能重要性。
